The N-terminal domain (1–318 amino acids) of mouse NFκB (p65) has been purified to homogeneity from the soluble fraction of Escherichia coli cells expressing this protein. Its complex with a full-length iκB-α (MAD3, 1–317 amino acids) molecule was generated by binding the E. coli-derived iκB-α to the purified NFκB and purifying the complex by sequential chromatography. The stoichiometry of NFκB to iκB in the complex was determined to be 2 to 1 by light scattering and SDS–polyacrylamide gel electrophoresis. The secondary structure of the NFκB (p65) determined by Fourier-transform infrared (FTIR) spectroscopy is in good agreement with that of the p50 in the crystal structure of the p50/DNA complex, indicating that no significant structural change in NFκB occurs upon binding of DNA. The FTIR spectrum of the NFκB/iκB complex indicates that its secondary structure is composed of 17% α-helix, 39% β-strand, 18% irregular structures, and 26% β-turns and loops. By comparing these data to the FTIR data for NFκB alone, it is concluded that the iκB (MAD3) in the complex contains 35% α-helix, 27% β-strand, 22% irregular structures, and 16% β-turns and loops. Circular dichroism (CD) analysis of a shorter form of iκB (pp40) indicates that it contains at least 20% α-helix and that the iκB subunit accounts for nearly all of the α-helix present in the NFκB/iκB complex, consistent with the FTIR results. The stabilities of NFκB, iκB, and their complex against heat-induced denaturation were investigated by following changes in CD signal. The results indicate that the thermal stability of iκB is enhanced upon the formation of the NFκB/iκB complex.